Clostridial neurotoxins with altered persistency

ABSTRACT

The invention relates to a polypeptide comprising:
         (a) a HC-domain or fragment thereof of the neurotoxic component of a clostridial toxin; and   (b) a first LC domain or fragment thereof of the neurotoxic component of a clostridial toxin; and   (c) at least one further LC domain or fragment thereof of the neurotoxic component of a clostridial toxin wherein the first and the at least one further LC domain may be the same or different from each other, and wherein each of said fragments of said first and of said at least one further LC domain still exhibits proteolytic activity.

FIELD OF THE INVENTION

The present invention relates to Clostridial neurotoxins, e.g.,botulinum neurotoxins, that are altered with regard to their proteinstructure in comparison to the corresponding wild-type neurotoxins. Saiddifference in protein structure results, inter alia, in a shiftedtime-period of activity, e.g. a prolonged activity or persistency.

BACKGROUND OF THE INVENTION

Chemodenervation refers to the use of an agent to prevent a nerve fromstimulating its target tissue, e.g. a muscle, a gland or another nerve.Chemodenervation is for example performed with phenol, ethyl alcohol, orbotulinum toxin. Chemodenervation is for example appropriate in patientswith localized spasticity in one or two large muscles or several smallmuscles. It may be used to alleviate symptoms such as muscle spasm andpain, and hyperreflexia.

Chemodenervating agents block neuromuscular transmission at theneuromuscular junction, causing paralysis or paresis of the affectedskeletal muscles. The term “paresis” is defined hereinunder as acondition typified by partial loss of movement, or impaired movement.This is accomplished either by acting presynaptically via the inhibitionof acetylcholine (ACh) synthesis or release, or by actingpostsynaptically at the acetylcholine receptor. Example of drugs thatact presynaptically are botulinum toxin, tetrodotoxin and tetanus toxin.

The term “chemodenervation” also encompasses all effects which directlyor indirectly are induced by the chemodenervating agent, therefore alsocomprising upstream, downstream or long-term effects of saidchemodenervating agent. Therefore, presynaptic effects are alsoencompassed as well as postsynaptic effects, tissue effects and/orindirect effects via spinal or afferent neurons.

One chemodenervating agent, botulinum toxin, although being one of themost toxic compounds known to date, has in the past been used for thetreatment of a large number of conditions and disorders, some of whichare described in e.g. PCT/EP 2007/005754. Furthermore, commercial formsof botulinum toxin type A based on the botulinum toxin A protein complexare available under the tradename BOTOX® (Clostridium botulinum toxintype A purified toxin complex,(900 kDa), Allergan Inc.) and under thetradename DYSPORT® (Clostridium botulinum type A toxin-haemagglutinincomplex: Ipsen Ltd.), respectively. A pharmaceutical composition basedon a higher purified toxin preparation and comprising the neurotoxiccomponent of botulinum toxin type A free of complexing proteins inisolated form is commercially available in Germany from MerzPharmaceuticals GmbH under the tradename XEOMIN® (inocbotulinumtoxinA;Clostridium botulinum type A neurotoxin (150 kDa), free of complexingproteins).

The anaerobic, Gram-positive bacterium Clostridium botulinum produces apotent polypeptide neurotoxin, botulinum toxin, which causes aneuroparalytic illness in humans and animals referred to as botulism.The spores of Clostridium botulinum are found in soil and can grow inimproperly sterilized and sealed food containers of home basedcanneries, which are the cause of many of the cases of botulism.Botulinum toxin A (BoNT/A) is the most lethal natural biological agentknown to man. About 50 picograms of botulinum toxin (purified neurotoxincomplex) serotype A is a LD₅₀ in mice. However, despite its toxiceffects, botulinum toxin complex as well as the pure neurotoxin havebeen used as a therapeutic agent in a large number of diseases.

Botulinum toxins are released from lysed Clostridium cultures generallyin the form of a protein responsible for the toxic properties of thebotulinum toxin (the neurotoxic component) in association with otherbacterial proteins (the non-toxic “complexing proteins” or “clostridialproteins”), which together form a toxin complex also designated“botulinum toxin complex”. The botulinum toxin complex is metastable innature, since its stability appears to depend on various factors such ase.g. salt concentration and/or pH. The molecular weight of the complexmay vary from about 300,000 to about 900,000 Da i.e. from 300 kDa toabout 900 kDa. The complexing proteins are, for example, varioushemagglutinins. The proteins of this toxin complex are not toxicthemselves but are believed to provide stability to the neurotoxiccomponent and are responsible for oral toxicity in Botulinumintoxications. There are seven antigenically distinct serotypes ofbotulinum toxin, namely botulinum toxin A, B, C1, D, E, F and G.Wherever the botulinum toxin serotype A, B, C1, D, E, F or G arementioned, also known variants of the serotypes are encompassed, likeserotypes A1, A2, A3, A4 etc.

The component of clostridial toxins responsible for its high toxicity isthe neurotoxic component or protein (Mw≈150 kD, exact molecular weightdepending of the serotype). The several different serotypes differ intheir amino acid sequence, but possess all a similar structure: a lightchain (LC) of approximately 50 kDa and a heavy chain (HC) ofapproximately 100 kDa, which may be linked by one or more disulfidebonds (for a review see e.g. Simpson L L, Ann Rev Pharmacol Toxicol.2004; 44:167-93). The neurotoxic component of the botulinum toxincomplex is initially formed as a single polypeptide chain. In the caseof serotype A, for example, proteolytic processing of the polypeptideresults in an activated polypeptide in the form of a dichain polypeptideconsisting of a heavy chain and a light chain, which are linked by adisulfide bond. In humans, the heavy chain mediates binding topresynaptic cholinergic nerve terminals and internalization of the toxininto the cell. The light chain is believed to be responsible for thetoxic effects, acting as zinc-endopeptidase and cleaving specificproteins responsible for membrane fusion (SNARE complex) (see e.g.Montecucco C., Shiavo G., Rosetto O: The mechanism of action of tetanusand Botulinum neurotoxins. Arch Toxicol. 1996; 18 (Suppl.): 342-354)).

The term “botulinum toxin” as used throughout the present application,refers to the neurotoxic component devoid of any other clostridialproteins, but also to the “botulinum toxin complex”. The term “botulinumtoxin” is used herein in cases when no discrimination between the toxincomplex and the neurotoxic component is necessary or desired. “BoNT” or“NT” are common used abbreviations for botulinum neurotoxin orneurotoxin, respectively. The neurotoxic subunit of the botulinum toxincomplex is referred in this document as the “neurotoxic component” orthe“neurotoxic component free of complexing proteins”. The production ofthe neurotoxic component of botulinum toxin type A and B are described,for example, in the international patent application WO 00/74703.

The several serotypes differ by their duration of therapeutic effect:The normal period of activity of botulinum toxin A drugs is, if injectedintramuscular in humans, between 3 and 4 months. In single cases theperiod can even extend to more than 12 months. During the treatment ofsweat glands, an activity of even 27 months has been reported (BusharaK., botulinum toxin and rhinorrhea, Otolaryngol. Head. Neck. Surg.,1996; 114(3):507 and The Laryngoscope 109: 1344 1346:1999). The periodof activity for botulinum toxin type C1 is comparable with the period ofactivity of botulinum toxin A (Eleopra et al., 1997 & 2002).Surprisingly the period of action is much shorter in rodents (e.g. mice)as compared to humans: Approximately 1-2 months for botulinum toxin A,21 days for botulinum toxin B and only 4 days for botulinum toxin E(DePaiva et al., 1999, Juradinski et al., 2001).

Foran et al. analyzed in 2003 the time period of action in vitro oncerebellum-neurons of rats and found half-times of the inhibition ofglutamate exocytosis for botulinum toxin A of more than 31 days; forbotulinum toxin type C1 of more than 25 days; for botulinum toxin type Bof approximately 10 days; for botulinum toxin type F of approximately 2days and for botulinum toxin type E of only 0.8 days.

The time period of activity of botulinum toxin type A during e.g. thetreatment of dystonias (e.g. Torticollis, Blepharospasmus) in humans isbetween 3 to 4 months. After this period the patient has to receiveanother injection of a botulinum toxin-containing drug. It would be ofgreat advantage for the patient to prolong the time period of action ofthe neurotoxin. In doing so, the number of necessary injections per yearwould be reduced as well as the overall amount of clostridial proteins.This again would reduce the risk of the production of antibodies againstthe foreign protein. Therefore, the provision of a botulinum toxin withprolonged persistency would be desirable.

However, not always long-term paralyzation is desired. For example incertain cosmetical treatments sometimes only temporal “fine-adjustments”are required. To achieve a reduction of persistency the physician wasrestricted by the prior art methods to either the reduction of volume orthe switch of serotype. These techniques proved to result inunsatisfying results and required profound knowledge both of theactivity kinetics as well as the antigenicity of the differentneurotoxin serotypes. Therefore, the provision of a neurotoxin with a“built-in” adjustment of persistency would be a major improvement.

US 2003/0219462, EP1849801 and WO 02/08268. disclose modified Botulinumtoxins with added leucine- or tyrosine-based motifs to the nativeneurotoxin.

The idea for these alterations is based on the observation that certainleucine- or tyrosine-based motifs enable the localization of the lightchain of the neurotoxic component of certain subtypes to the innermembrane of the target cell. This mechanism was hypothesized to changethe persistency of certain light chains. Until now, however, the authorsfailed to provide any evidence for such an effect and newer experimentssuggest that the whole hypothesis is inaccurate.

Furthermore, even if in certain cases an addition of motifs would leadto a membrane localization, such an approach is not applicable tomodifications of Botulinum toxin A. This is because the native lightchain of Botulinum toxin type A is already localized to the innercell-membrane, therefore an additional tethering to the membrane doesnot provide any additional benefit.

Therefore the present invention followed a different path. As it hasbeen found as disclosed in this application, the addition of a secondlight chain to the neurotoxin, which still possesses its proteolyticactivity, leads to an alteration of the time period of activity.Depending on the combination of serotypes used, the time-period can beprolonged, allowing for the production of custom-tailored neurotoxins.It is envisaged to provide the physician with a range of neurotoxins,whose serotype is independent of their persistency, allowing for a morestandardized treatment.

SUMMARY OF THE INVENTION

The present invention relates to clostridial neurotoxins, in oneembodiment botulinum toxins, with increased or prolonged activity, i.e.persistency. Thus, in a first aspect the present application relates toa polypeptide comprising:

-   -   (a) a HC-domain or fragment thereof of the neurotoxic component        of a clostridial toxin; and    -   (b) a first LC domain or fragment thereof of the neurotoxic        component of a clostridial toxin; and    -   (c) at least one further LC domain or fragment thereof of the        neurotoxic component of a clostridial toxin wherein the first        and the at least one further LC domain may be the same or        different from each other, and wherein each of said fragments of        said first and of said at least one further LC domain still        exhibits proteolytic activity.

In one embodiment the units and/or domains are connected via a bond, apeptide-linker, a chemical linker, a disulfide bond, or via acombination of two or more thereof.

In one embodiment the amino acid sequence of said LC and/or HC domain isat least 50% identical to the amino acid sequence of a neurotoxiccomponent of botulinum toxin of serotype A, B, C, D, E, F or G.

In another embodiment the amino acid sequence of said LC and/or HCdomain is at least 50% identical to the amino acid sequence of tetanustoxin (tetanospasmin).

In one embodiment the first and/or the second LC domain and/or the HCdomain comprise at least one modification.

In one embodiment the modification is a mutation, in another embodimenta deletion, in yet another embodiment an insertion, in yet anotherembodiment an addition or in yet another embodiment an amino acidexchange or in a further embodiment a combination of two or morethereof.

In one embodiment, the invention relates to a polypeptide, wherein, theganglioside-binding domain and/or the protein receptor binding-domain ofthe neurotoxin is modified such as to enhance the binding capacitycompared to the wild-type neurotoxin from which the HC domain isderived.

In one embodiment the polypeptide is one selected from the groupconsisting of:

-   -   LCBoNT/A-LCBoNT/A-HCBoNT/A, LCBoNT/C-LCBoNT/A-HCBoNT/A,        LCBoNT/B-LCBoNT/A-HCBoNT/A, LCBoNT/A-LCBoNT/C-HCBoNT/C,        LCBoNT/C-LCBoNT/C-HCBoNT/C, LCBoNT/B-LCBoNT/C-HCBoNT/C and        LCTeNT-LCBoNT/A-HCBoNT/A.

In yet another embodiment the modification is a chemical modification,whereas the chemical modification may be selected from the groupcomprising a phosphorylation, a pegylation, a glycosylation, aphosphorylation, a sulfatation, a methylation, an acetylation, alipidation, a hydroxylation, an amidation or in a further embodiment acombination of two or more thereof. In a further embodiment thelipidation may be a myristoylation, plamitoylation, isoprenylation orlinkage of glucosyl-phophatidylinositol or in a further embodiment acombination of two or more thereof.

The invention also discloses an antibody specific for any of the abovementioned polypeptides.

The invention also discloses a nucleic acid encoding for any of theabove mentioned polypeptides. The invention discloses furthermore avector comprising said nucleic acid or fragments thereof. A host cellcomprising said nucleic acid or said vector is also disclosed herein.

The invention also discloses a method for producing a polypeptidecomprising the steps of cultivating the host cell as mentioned before,producing and purifying said polypeptide encoded by said nucleic acid orvector and, optionally, formulating said polypeptide in a pharmaceuticalcomposition.

The invention furthermore discloses a composition comprising the abovementioned polypeptide or the polypeptide obtainable by the abovementioned method. The invention also discloses said composition furthercomprising a pharmaceutically acceptable carrier. In another embodimentthe composition further comprises a pH buffer, an excipient, acryoprotectant, a preservative, an analgesic, a stabilizer or anycombination thereof. In one embodiment the composition is provided as alyophilisate. In another embodiment the composition is provided as asolution.

The invention also discloses said compositions for use in a therapeutictreatment.

The invention also discloses the use of said compositions for themanufacture of a medicament for therapeutic treatment.

In one embodiment said therapeutic treatment comprises treatment offocal dystonia, spasticity or a condition which can be treated bysuppressing secretion.

The invention also discloses the use of said composition for cosmetictreatment. Within such cosmetic treatment, it may very well be that inparticular mammals are treated who psychologically suffer from thecondition, e.g. wrinkles, or glabella frown line, that is to be treated.

DETAILED DESCRIPTION OF THE INVENTION

The present invention relates to:

A polypeptide comprising:

-   -   (a) a HC domain of the neurotoxic component or fragment thereof        of a clostridial toxin; and    -   (b) a first LC domain or fragment thereof, and    -   (c) at least one further LC domain or fragment thereof, wherein        the first and second LC domain may be the same or different from        each other

In particular the invention relates to a polypeptide comprising:

-   -   (a) a HC-domain or fragment thereof of the neurotoxic component        of a clostridial toxin; and    -   (b) a first LC domain or fragment thereof of the neurotoxic        component of a clostridial toxin; and    -   (c) at least one further LC domain or fragment thereof of the        neurotoxic component of a clostridial toxin wherein the first        and the at least one further LC domain may be the same or        different from each other, and wherein each of said fragments of        said first and of said at least one further LC domain still        exhibits proteolytic activity.

Surprisingly, it has been found that the addition of one or more(further) light chains (LC) to a polypeptide comprising at least oneheavy chain (HC) and at least one light chain (LC) of the neurotoxiccomponent of clostridial neurotoxins, as defined above, results in apolypeptide with increased i.e. prolonged persistency of the toxinactivity as compared to the wild-type toxin.

Without being bound to theory it is hypothesized that, after binding tothe cell surface, both light chains are translocated into the cell,increasing the concentration of proteolytic active proteins in the cell,thereby both increasing the activity as well as the persistency of theneurotoxin.

That is, if a long persistency of the polypeptide of the invention isdesired, the artisan is instructed to use certain combinations of HC andLC domains, e.g. LC domains derived from serotypes with longpersistency. In other embodiments a shorter persistency is achieved bycombining certain other LC domains or fragments, e.g. such derived fromserotypes with shorter persistency.

The term “persistency” as used herein describes the time period ofaction of a neurotoxic component. In general this is the time perioduntil the active agent shows only half of its activity compared to itsstarting activity. Therefore, the term “persistency” can be usedsynonymously with the term “half-life of activity” or the term“half-life of metabolic stability” which defines the time point wherejust one half of the starting protein-concentration is active due tometabolic processes, i.e. the half-life until the protein ismetabolized. Since the half-life of the protein correlates with theduration of the therapeutic effect, the term “persistency” alsoindirectly encompasses the time duration of interference or influencecaused by a neurotoxic component with a cellular function.

The skilled person knows various assays for determining persistency.According to the teaching of the present invention, persistency may bedetermined with a mouse running assay (Keller J E., 2006, Neuroscience.139(2):629-37). This assay allows correlating persistency with movementactivity. Alternatively, persistency may be determined with a SNAP-25cleavage assay, which allows correlating proteolytic activity withpersistency. The effect of an increased persistency is fulfilled, if anincrease in persistency can be determined in one of the assays describedabove, wherein the SNAP-25 cleavage assay is preferred.

The term increased persistency and prolonged persistency are used hereinexchangeable.

For determining the impact of an additional LC chain, or LC chainfragment, on the polypeptide of the invention with regard topersistency, the polypeptide of the invention is compared to acorresponding polypeptide lacking said additional (further) LC chain.This may, for example, be a polypeptide of the invention from which theadditional LC chain has been deleted. Any of the persistency assaysknown to the person skilled in the art may be used for determiningpersistency. In one embodiment, persistency is determined as describedherein above or in the examples illustrating the invention.

In another embodiment the prolongation of the persistency of shortacting neurotoxin serotypes is envisaged. For example the persistency ofserotype E can be prolonged by adding light chains of longer activeserotypes like for example serotype A, thereby creating a neurotoxinwith a similar persistency as the wild-type botulinum toxin A. Sincemost of the antigenic epitopes of the neurotoxin are situated on theheavy chain subunit, this modification can be used to apply a neurotoxinin patients which have developed an immune response against a certainserotype. Thereby combining the advantage of providing a differentserotype by maintaining the previous time period of activity.

As indicated above, the terms “HC-domain” and “LC-domain” refer to theheavy chain respectively light chain of the neurotoxic component of theneurotoxin either of wild-type or recombinant origin. Furthermore, insome embodiments the HC and/or LC domains are derived from differentserotypes and/or different toxins. Within this definition also fragmentsof the light chain and heavy chain are encompassed. The HC- andLC-domain can additionally be further subdivided into sub-domains.

The term “further LC domain or fragment” thereof, as used herein refersto a one or more, e.g. second LC domain. According to the teaching ofthe present invention, the polypeptide of the invention may containfurther additional LC domains or fragments thereof. For example, thepolypeptide of the invention may comprise the HC domain of theneurotoxic component of a clostridial toxin and a first LC domain orfragment thereof and a second LC domain or fragment thereof and a thirdLC domain or fragment thereof.

In one embodiment, said fragment of said first and said further LCdomain exhibits the proteolytic activity of the wild type LC.

To achieve said effect of increased persistency no additional leucine-or tyrosine-based motif (as disclosed in US 2003/0219462, EP1849801 andWO 02/08268) is neither needed nor desired. Therefore, in oneembodiment, the additional light chain does not possess any of saidmotifs.

In one embodiment the modified neurotoxin does not contain aleucin-based motif comprising seven amino acids, wherein the first fiveamino acids starting from the amino-terminal of the leucine-based motifform a “quintet of amino acids” and the following two amino acids form a“dublet of amino acids” and wherein the quintet of amino acids comprisesat least one amino acid selected from a group consisting of glutamateand aspartate; and the duplet of amino acids comprises at least oneamino acid selected from from a group consisting of isoleucine andleucine.

In another embodiment the modified neurotoxin does not contain any ofthe sequences FEFYKLL (SEQ ID NO:1), EEKRAIL (SEQ ID NO:2), EEKMAIL (SEQID NO:3), SERDVLL (SEQ ID NO:4), VDTQVLL (SEQ ID NO:5), AEVQALL (SEQ IDNO:6), SDKQNLL (SEQ ID NO:7), SDRQNLI (SEQ ID NO:8), ADTQVLM (SEQ IDNO:9), SDKQTLL (SEQ ID NO:10), SQIKRLL (SEQ ID NO:11), ADTQALL (SEQ IDNO:12) and NEQSPLL (SEQ ID NO:13).

The term “the same as” used herein refers to an LC domain with identicalamino acid sequence, i.e. with 100% amino acid sequence identity.Therefore, e.g. a second LC domain, as used herein, which is “the same”means it is identical in amino acid sequence to said first LC domain. Onthe other hand, a second LC domain which is “different” refers to asecond LC domain which has a sequence identity of less than 100%, i.e.for example 99.95% or less compared to the first LC domain. A “differentLC domain” is an LC domain of a different serotype or an LC domain withan amino acid sequence that is different from the first LC domain, e.g.one with an amino acid substitution. Another example of a different LCdomain is an LC domain with a truncation at the N- or C-terminus or withan internal deletion. Yet another example of a different LC domain is anLC domain with a chemical modification. A “different LC domain” maytherefore be derived from the same or a different serotype, respectivelycompared to the first LC domain. The above also applies to a “third” orany other additional LC domain.

In one embodiment the serotypes of all HC and LC domains are frombotulinum toxin type A, in another embodiment the second light chain isof serotype C1. However, it is clear to the person skilled in the art,that all possible combinations of serotypes A, B, C1, D, E, F and G arecovered by this application and the skilled person is able to choose anappropriate combination based on the published persistency of thedifferent serotypes. Neither the combination of serotypes nor the numberof used heavy and light chains is restricted by this invention.Therefore, in another embodiment longer fusion proteins, i.e. fusionprotein with more than three subunits are envisaged, e.g.protein-concatemers comprising three, four, five, six, seven, eight,nine or ten LC domains.

The HC and LC domains of for example the neurotoxin A of C. botulinumcomprise different subdomains. The HC domain, for example comprisesthree sub-domains, i.e. amino-terminal 50 kDa translocation sub-domainHC_(N) with the subsequent 25 kDa HC_(CN)-sub-domain and the 25 kDaHC_(CC)-sub-domain located carboxy-terminally. Taken together, theHC_(N)-, HC_(CN) and HC_(CC)-domains are designated as HC-domain.

The respective amino acid ranges of the respective domains are shown forthe different BoNT/A serotypes and its variations in table 1.

TABLE 1 Database accession numbers of the amino acid sequences ofbotulinum neurotoxin A subtypes 1-4 and amino acid ranges of therespective domains. HC BoNT/A Amino HC_(C) subtype Genbank # acids #HC_(N) HC_(CN) HC_(CC) A1 AAA23262 1296 449-866 867-1091 1092-1296AAM75961 AAQ06331 BTCLAB ABP48105 ABP48106 ABO68834 ABO68833 ABD65472AAQ06331 P10845 1296 449-866 867-1091 1092-1296 CAA36289 1296 449-866867-1091 1092-1296 A2 CAA51824 1296 449-866 867-1091 1092-1296 I40645Q45894 AAX53156 ABC26002 1296 449-866 867-1091 1092-1296 A3 ABA290171292 445-862 863-1087 1088-1292 A4 ABA29018 1296 449-866 867-10911092-1296

The term “fragment of the LC domain”, as used herein, refers to afragment of the LC domain with biological activity. As used herein, afragment with biological activity is a fragment which (still) exhibitsthe proteolytic activity preferably of the wild-type LC, i.e. which iscapable of cleaving a polypeptide of the SNARE complex such as e.g.syntaxin, SNAP-25 or synaptobrevin. Accordingly, biological activity maybe tested e.g. by a SNAP-25 protease assay, LD₅₀-Assay, HDA-Assay, andthe like. Therefore, any LC-domain, which shows proteolytic activity ofmore than 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90% and up to 100% ofthe corresponding wild-type LC-domain in a SNAP-25 assay is considered“biological active” or “to exhibit proteolytic activity” within thescope of this invention.

A suitable SNAP-25 assay is for example the “GFP-SNAP25 fluorescencerelease assay” (WO/2006/020748) or the “improved SNAP25 endopeptidaseimmuno-assay” (Jones et al., Journal of Immunological Methods, Volume329, Issues 1-2, 1 Jan. 2008, Pages 92-101).

A “fragment of the HC domain”, as used herein, refers to a fragment ofthe HC domain with biological activity. More specific, this is afragment which is still capable of binding to the native HC domainreceptor, from which it is derived. Moreover, said fragment is also afragment capable of translocating an LC domain attached to it.

Fragments, therefore, are e.g. polypeptides of which 1, 2, 3, 5, or upto 10, 50, or 100 amino acids have been deleted. Wherein the deletioncan be a truncation at the C- or N-terminus or an internal deletion.

In some embodiments the HC and/or LC domains are additionally modifiedby for example a mutation, a deletion, an insertion, an addition or anamino acid exchange. In further embodiments the HC and/or LC areadditionally be chemically modified, for example by a phosphorylation, apegylation, a glycosylation, a phosphorylation, a sulfatation, amethylation, an acetylation, a lipidation (myristoylation,palmitoylation, isoprenylation, linkage ofglucosyl-phophatidylinositol), a hydroxylation, an amidation or anyother suitable modification. Additionally the ganglioside-binding domainand/or the binding-domain of the neurotoxin are in one embodimentmodified such as to enhance the binding capacity compared to thewild-type neurotoxin from which the HC domain is derived. In someembodiments, the HC and/or LC comprise a tag-sequence, i.e. anotheramino acid sequence, which allows a simplified purification procedure.

The term “purification method” encompasses all methods known in the artfor protein purification. Examples for purification methods forneurotoxins are the publications of DasGupta & Sathyamoorthy andWO2000074703 which are incorporated by reference herein. For furtherguidance of protein purification methods useful for the purification ofrecombinant neurotoxic components, reference is made to the documents ofWalker et al., 2002; Harris et al. 1989 and Scopes et al., 1994, whichare cited in the section “Literature” below.

The term “production of the polypeptide” encompasses all steps necessaryfor the production of the polypeptide, i.e. for example creation of theencoding nucleic acid, incorporation said nucleic acid into a vector,expression of the polypeptide in vitro and/or a host cell, modificationsof the polypeptide in vivo and/or in vivo, purification of thepolypeptide and/or production of a composition containing saidpolypeptide. Thereby the term “expression” or “gene expression” isdefined herein as the process by which the inheritable information in agene, such as the DNA sequence, is made into a functional gene product,such as protein or RNA.

In one embodiment it is envisaged to incorporate into the polypeptide ofthe present invention additional receptor binding sites to provide aneurotoxin which possesses, besides an increased persistency, additionalcharacteristics allowing for new applications, e.g. a neurotoxin withcell-specific binding sites suitable for example for the treatment ofallergies or pain (WO 2007/13839). Alternatively, the native bindingsite located within the HC domain may be altered in order to target thepolypeptide of the present invention to specific cell types. In a moreparticular example, the HC domain of the present invention.

In one embodiment the second light chain is connected to the N-terminusof the first light chain. This connection can be either directly via abond or indirectly via a linker. In general the bonding between thedomains can be achieved via any entity suitable to hold the differentsubunits together, comprising, besides others, direct linkage or linkagevia a peptide-linker, via a chemical linker or via a disulfide bond.Said bond may be a cleavable or a non-cleavable bond. A cleavable bondis a bond which is cleavable by e.g. a sequence specific protease. Anon-cleavable bond is a bond which is stable after cellular uptake, inother words, several LC domains connected by a non-cleavable bond remainbound to each other, even after translocation into the cytoplasm.

The terms “bond”, “bonds” or “bonding” describe any possibility toconnect the different polypeptide chains with each other. In oneembodiment said bond is a chemical bond, e.g. covalent bond (e.g.disulfid-bond), polar covalent bond, ionic bond, coordinate covalentbond, bent bonds, 3c-2e and 3c-4e bonds, one- and three-electron bonds,aromatic bond, metallic bond, intermolecular bonding, permanent dipoleto permanent dipole bonding, hydrogen bond, instantaneous dipole toinduced dipole (van der Waals) bonding and/or cation-pi interaction. Asmentioned above, this definition encompasses direct bonds as well asindirect bonds via chemical linkers.

A “chemical linker” is defined herein as an molecule entity produced bychemical means, which is suitable to connect the different subunits ofthe polypeptide of the present invention. These chemical linkage can beachieved for example by bifunctional agents known in the art. In anotherembodiment the chemical linkage is achieved by di-sulfid bonds, similarto the connection between heavy and light chain in the wildtype. In yetanother embodiment the introduction of a disulfide bond is achieved byintroducing a cysteine containing sequence of the heavy chain (e.g. aa449-459 of BoNT/A) into the light chain. Further non-limiting examplesfor such chemical linkers are carboxylic acids, ethoxylated polyhydricalcohol, polyvinyl pyrrolidone, polyethylene glycol etc.

A “peptide linker” is defined herein as a peptide of 1, 2, 3, 4, 5, 10,20, 30, 40, 50, or up to 100 amino acids length, which connects thedifferent subunits of the polypeptide of the present invention with eachother. In one embodiment said peptide-linker comprises at least twocysteines. In another embodiment the linker comprises one, two, three,four, five, six, seven, eight, nine, ten or up to 20 histidines, inanother embodiment the linker is a protease cleavage site. In a furtherembodiment the linker enables the production of the full fusion proteinby recombinant methods.

In another embodiment a protease cleavage site can be introduced betweenthe first and the second light chain, e.g. a site which can be cut by E.coli proteases as they are listed for example in DE102005002978 butwithout the restriction to these proteases. In another embodiment theprotease cleavage site is any of the recognition sites for either serineproteases (e.g. chymotrypsin, trypsin, elastase, subtilisin), threonineproteases, cysteine proteases (e.g. papain, cathepsin, caspase,calpain), aspartic acid proteases (e.g. HIV-protease, chymosin, renin,cathepsin, pepsin, plasmepsin), metalloproteases or glutamic acidproteases or any combination thereof.

The person skilled in the art will understand that this invention is notonly suitable for the usage of wild-type heavy and light chain(s), butthat also recombinant peptides and/or hybrid neurotoxic components areencompassed by this invention. Therefore, in one embodiment a fusionprotein of at least one heavy chain, at least a first light chain and atleast a second light chain is envisaged, wherein at least one, some orall of the used domains are produced recombinantly, in anotherembodiment hybrid peptides are used, i.e. peptides composed ofsub-domains from different serotypes (e.g. a heavy chain comprising abinding and a translocation domain of a different serotypes or even adifferent toxin, e.g. tetanus toxin, cholera toxin or pertussis toxin).

In another embodiment the light chain(s) of other clostridial toxins,e.g. Clostridium bifermentans, Clostridium botulinum of a differentserotype, Clostridium difficile, Clostridium histolyticum, Clostridiumkluyveri, Clostridium novyi, Clostridium oedematiens, Clostridiumperfringens, Clostridium ramosum, Clostridium sporogenes, Clostridiumtetani, Clostridium tertium or Clostridium welchii can be used, e.g. inone embodiment tetanus toxin (also called tetanospasmin or spasmogenictoxin) is used as well as any variations and serotype of the differenttoxins. In addition the cell binding part of the heavy chain can beexchanged with a polypeptide sequence which endows the fusion proteinwith another targeting domain i.e. another cell specificity (e.g. WO2007/13839). Furthermore yet another embodiment of the invention makesuse of heavy and light chains, which have been altered by molecular orbiochemical methods, more preferably deletions, insertions, amino acidexchange or elongation.

In one embodiment the serotype of the translocation sub-domain of HC(i.e. the N-terminal part of the heavy chain) is the same serotype likethe one of the first LC.

In one embodiment the SNARE-complex cleaving ability of the LC-domain isof major interest. Therefore, in one embodiment one of the LC-domains ofthe fusion protein is exchanged by the IGA protease from Neisseriagonorrhoeae, which possesses SNARE-complex cleaving ability as well.

In further embodiments the present invention also refers to neurotoxinswhich are chemically modified, e.g. by pegylation, glycosylation,sulfatation, phosphorylation or any other modification, in particular ofone or more surface or solvent exposed amino acid(s).

Furthermore, in another embodiment, the neurotoxin possesses atag-sequence to allow for simplified purification methods. Such knownlabelling methods make use of small molecules or peptides, e.g. biotin,streptavidin, strep-tag, His-tag, antigens, antibody-fragments etc.which are covalently or non covalently bound to the polypeptide of thepresent invention and enable the purification viaaffinity-chromatgraphy, beads or other separation methods.

As stated above in some embodiments the fusion protein containsrecombinant domains or is produced recombinantly in full. DNA-sequencesof all heavy and light chains of all serotypes of botulinum toxin areavailable from public databases Therefore, it is envisaged to constructvectors carrying the desired genes for the heavy and light chains byrelying on these database information. The vector then is expressed ine.g. E. coli to produce a fusion protein. In another embodiment thevector can be expressed in other expression systems, like for exampleyeast, insect cells or CHO-cells. In another embodiment the proteindomains are produced separately and then connected later by chemicalmethods. The resulting protein is then isolated by known methods ofprotein purification, then, if necessary, further processed (e.g.cleavage, chemical linkage or treatment) and used as an active agent ina pharmaceutical formulation.

In one embodiment the modified neurotoxin is additionally modified toalter (i.e. increase and decrease) its binding affinity to its receptor.Binding affinity may be determined in comparison to a native neurotoxin,i.e. a neurotoxin derived from C. botulinum and having a wild-type aminoacid sequence. Alternatively, binding assays may be performed with afragment of said neurotoxin. Preferably said neurotoxin is obtainablefrom C. botulinum. An increased affinity means that the neurotoxinaccording to the invention has a lower dissociation constant incomparison to the non-modified neurotoxin. Preferably, the nativeneurotoxin is botulinum neurotoxin of serotype A including any subtypeA, which is defined in detail below. A recombinantly produced botulinumneurotoxin of serotype A, whose amino acid sequence is identical to abotulinum neurotoxin obtained from C. botulinum, behavespharmacologically identical or similar to the native botulinumneurotoxin obtained from C. botulinum. Such a recombinant neurotoxin maybe produced in e.g. E. coli and is commonly referred to as “recombinantbotulinum neurotoxin”. Binding assays may be performed with a neurotoxinisolated from C. botulinum or a neurotoxin obtained by recombinantprotein expression. Preferably, the polypeptide, the active fragment orderivative according to the present invention binds specifically toplasma membrane associated molecules, transmembrane proteins, synapticvesicle proteins, a protein of the synaptotagmin family or the synapticvesicle glycoproteins 2 (SV2), preferably synaptotagmin I and/orsynaptotagmin II and/or SV2A, SV2B or SV2C, particularly preferred humansynaptotagmin I and/or human synaptotagmin II and/or human SV2A, SV2B orSV2C. The binding is preferably determined in vitro. The skilled personknows various assays for determining binding affinities between a firstprotein (the neurotoxin) and a second protein (the receptor). Any suchassay may be useful for determining the effect of a mutation on receptorbinding. One such assay is a GST-pull-down-assay, which is preferred inaccordance with the teaching of the present invention. This assay isdescribed in the examples of the present invention. Surface plasmonresonance may also be used to study the binding affinity. Experimentalconditions therefore, are e.g. described in Yowler et al., Biochemistry43 (2004), 9725-9731. In addition, the binding affinity may be assessedusing isothermal microcalorimetry. In one embodiment theganglioside-binding domain and/or the protein receptor binding-domain ofthe neurotoxin is modified such as to enhance the binding capacitycompared to the wild-type neurotoxin from which the HC domain isderived. As reference it is referred to WO2006/027207 A1, WO 2006/114308A1 and PCT/EP2008/006151 (EP 07 014 785.5) which are fully incorporatedin this document.

In another embodiment, also isoforms, homologs, orthologs and paralogsof botulinum toxin are encompassed, which show at least 50%, at least60%, at least 70%, at least 80%, at least 90% and up to 60%, up to 70%,up to 80%, up to 90%, up to 100% sequence identity. The sequenceidentity can be calculated by any algorithm suitable to yield reliableresults, for example by using the FASTA algorithm (W. R. Pearson & D. J.Lipman PNAS (1988) 85:2444-2448). Sequence identity may be calculated bycomparing two polypeptides or two domains such as two LC domains orfragments thereof.

In one embodiment the polypeptide of the invention is one of thefollowing: LCBoNT/A-LCBoNT/A-HCBoNT/A, LCBoNT/C-LCBoNT/A-HCBoNT/A,LCBoNT/B-LCBoNT/A-HCBoNT/A, LCBoNT/A-LCBoNT/C—HCBoNT/C,LCBoNT/C-LCBoNT/C-HCBoNT/C, LCBoNT/B-LCBoNT/C—HCBoNT/C andLCTeNT-LCBoNT/A-HCBoNT/A.

Of these before mentioned modified neurotoxins especially the constructswith an additional light chain type A are to be mentioned, due to itsexcellent proteolytic activity and stability.

The invention also relates to an antibody able to specifically bind tothe polypeptide of the present invention.

The term “antibody” is used herein for any protein or polypeptide, whichis able to bind specific to the polypeptide of the invention (e.g. aminoacids, primary, secondary or tertiary structure elements, epitopes,fragments, etc.). Examples for antibodies are the gamma-globulins IgA,IgD, IgE, IgG and IgM, fragments thereof, modified versions thereof,etc.; also any gene-product of the V, D, J genes, T-cell-receptors,B-cell-receptors, etc. Also included are single chain antibodies ormodified antibodies such as humanized antibodies. Since antigens arejust defined by its ability to be bound by an antibody, they represent avery heterogeneous group. Examples for “antigens” are proteins;oligopepeptides; sugars, lipids, lipopolysaccharides, cellular, viral orbacterial surface molecules; macromolecules, etc. The term “specific”describes a binding affinity high enough to differentiate betweendifferent structural patterns, i.e. the difference between the affinityfor the antigen should be at least 10 times, 20 times, 10² times, 10³times, 10⁴ times, 10⁵ times, 10⁶ times, 10⁷ times, 10⁸ times, up to 10⁹times higher then the affinity to a reference structure, which is notthe antigen or epitope. In one embodiment, said reference structure isnot the neurotoxin of serotype A to G. The antibody of the presentinvention is specific for the polypeptide of the present invention. Inone embodiment it does not bind to the wild-type neurotoxins (i.e.serotypes A to G) and/or other neurotoxins and/or neurotoxin fragmentsknown in the art, in another embodiment said antibody binds with muchreduced affinity to the wild-type neurotoxins and/or other neurotoxinsknown in the art, whereas the difference in affinity high enough, thatthe antibody is still suitable for purification, toxin inactivationand/or detection methods. Examples for antibodies of this invention aresuch antibodies, which recognize the additional LC-domain(s) and/ormodifications of the additional LC-domain(s).

Such antibodies may be produced by the known method in the art.Furthermore several methods are known how to positively and negativelyselect for antibodies, which recognize the polypeptide of the inventionbut not (or to a much lesser degree) known neurotoxins and/or fragments.As example reference is made to the documents WO2005/063817,WO2003/029458 and WO2002/086096 which are fully incorporatedhereinunder.

Said antibody is in one embodiment suitable for purification, toxininactivation and/or detection methods. Examples for the application ofsuch antibodies are for example HDA (hemidiaphragma assay),immunoprecipitation, affinity-chromatography, western-blots, etc.

The invention also encompasses nucleic acids encoding the polypeptide ofthe invention. In one embodiment said nucleic acid contains additionalsequences known in the art like e.g. promotors, enhancers, bacterialelements, IRES-regions, terminal capping structures etc. This nucleicacid molecule can be hnRNA, mRNA, RNA, DNA, PNA, LNA, and/or modifiednucleic acid molecules etc. The nucleic acid can be circular, linear orintegrated into a genome. Also DNA-concatemers coding for fusionproteins comprising three, four, five, six, seven, eight, nine or ten LCdomains are encompassed.

The invention also encompasses a vector suitable for in vitro and/or invivo expression of the polypeptide of the present invention. Whereas invivo the vector can be transient and/or stable expressed. In oneembodiment the vector furthermore comprises regulatory elements and/orselection markers. Said vector in one embodiment is based on virusorigin, in another embodiment of phage origin, in yet another embodimentof bacterial origin.

The invention also encompasses prokaryotic and/or eukaryotic host cellssuitable to express said vector and in particular the polypeptide of theinvention. In one embodiment said host cell is of clostridial origin, inanother embodiment said host cell is derived from standard cells forrecombinant expression, e.g. E. coli, etc. In one embodiment, thepolypeptide is modified inside the host cell (i.e. glycosylated,phosphorylated, processed by proteases, etc.), Therefore, both thepre-polypeptide, any intermediate protein product as well as the finalpolypeptide are encompassed by this invention.

The polypeptide of the invention may be part of a composition or apharmaceutical composition. A “pharmaceutical composition” is aformulation in which an active ingredient for use as a medicament or adiagnostic is contained or comprised. Such pharmaceutical compositionmay be suitable for diagnostic or therapeutic administration (i.e. byintramuscular or subcutaneous injection) to a human patient.

This pharmaceutical composition to be used herein may comprise thepolypeptide of the invention (i.e. the modified neurotoxic component) asthe sole active component or may contain additional pharmaceuticallyactive components e.g. a hyaluronic acid or a polyvinylpyrrolidone or apolyethleneglycol, such composition being optionally pH stabilized by asuitable pH buffer, in particular by a sodium acetate buffer, and/or acryoprotectant polyalcohol.

Within one embodiment of the present invention it is envisaged that thepharmaceutical formulation contains no proteins found in the botulinumtoxin complex other than the neurotoxic component which is part of thepolypeptide of the present invention. The precursor of the polypeptideof the present invention may be cleaved or uncleaved, however, within anembodiment of particular interest the precursor has been cleaved intothe heavy and the light chains. As pointed out above, the polypeptidesmay be of wild-type sequence or may be modified at one or more residues.Modification comprises chemical modification e.g. by glycosylation,acetylation, acylation or the like, which may be beneficial e.g. to theuptake or stability of the polypeptide. The polypeptide chain of thepolypeptide of the invention may, however, alternatively or additionallybe modified by addition, substitution or deletion of one or more aminoacid residues.

In one embodiment, the polypeptide of the invention has a biologicalactivity of 10 to 500 LD₅₀ units per ng polypeptide of the invention, asdetermined in a mouse LD₅₀ assay. In another embodiment, the polypeptideof the invention has a biological activity of about 150 LD₅₀ units pernanogram. Generally, the pharmaceutical composition of the presentinvention comprises the polypeptide of the invention in a quantity ofabout 6 pg to about 30 ng.

A pharmaceutical composition comprising the neurotoxic component ofbotulinum toxin type A in isolated form is commercially available inGermany from Merz Pharmaceuticals GmbH under the trademark XEOMIN®. Theproduction of the neurotoxic component of botulinum toxin type A and Bare described, for example, in the international patent applications WO00/74703 and WO 2006/133818. The skilled person can adapt saidcompositions to the polypeptide of the invention referred herein.

In one embodiment, said composition is a reconstituted solution of thepolypeptide of the invention. In another embodiment the compositionfurther comprises sucrose or human serum albumin or both, still anotherembodiment the ratio of human serum albumin to sucrose is about 1:5. Inanother embodiment, said human serum albumin is recombinant human serumalbumin. Alternatively, said composition is free of mammalian derivedproteins such as human serum albumin. Any such solution may providesufficient neurotoxin stability by replacing serum albumin with othernon-proteinaceous stabilizers (infra).

Within the present patent application, the use of a medicament based onthe modified neurotoxic component mentioned above can be used.

With regard to the composition and dosing of the medicament on the basisof botulinum toxin, and in regard to the composition, dosing andfrequency of administration of the medicament on the basis of theneurotoxic component of botulinum toxin, reference is made toPCT/EP2007/005754.

The pharmaceutical composition may be lyophilized or vacuum dried,reconstituted, or may prevail in solution. When reconstituted, in oneembodiment the reconstituted solution is prepared adding sterilephysiological saline (0.9% NaCl).

Such composition may comprise additional excipients. The term“excipient” refers to a substance present in a pharmaceuticalcomposition other than the active pharmaceutical ingredient present inthe pharmaceutical composition. An excipient can be a buffer, carrier,antiadherent, analgesic, binder, disintegrant, filler, diluent,preservative, vehicle, cyclodextrin and/or bulking agent such asalbumin, gelatin, collagen, sodium chloride, preservative,cryoprotectant and/or stabilizer.

A “pH buffer” refers to a chemical substance being capable to adjust thepH value of a composition, solution and the like to a certain value orto a certain pH range. In one embodiment this pH range can be between pH5 to pH 8, in another embodiment pH 7 to pH 8, in yet another embodiment7.2 to 7.6, and in yet a further embodiment a pH of 7.4. In anotherembodiment the pharmaceutical composition has a pH of between about 4and 7.5 when reconstituted or upon injection, in yet another embodimentabout pH 6.8 and pH 7.6 and in a further embodiment between pH 7.4 andpH 7.6.

In one embodiment the composition also contains a 1-100 mM, in anotherembodiment 10 mM sodium acetate buffer.

The pH ranges given mentioned above are only typical examples and theactual pH may include any interval between the numerical values givenabove. Suitable buffers which are in accordance with the teaching of thepresent invention are e.g. sodium-phosphate buffer, sodium-acetatebuffer, TRIS buffer or any buffer, which is suitable to buffer withinthe above pH-ranges.

“Stabilizing”, “stabilizes” or “stabilization” means that the activeingredient, i.e., the polypeptide of the invention in a reconstituted oraqueous solution pharmaceutical composition has greater than about 20%,30%, 40%, 50%, 60%, 70%, 80%, 90%, and up to about 100% of the toxicitythat the biologically active polypeptide of the invention had prior tobeing incorporated into the pharmaceutical composition.

Examples of such stabilizers are gelatin or albumin, in one embodimentof human origin or obtained from a recombinant source. Proteins fromnon-human or non-animal sources are also included. The stabilizers maybe modified by chemical means or by recombinant genetics. In oneembodiment of the present invention, it is envisaged to use alcohols,e.g., inositol, mannitol, as cryoprotectant excipients to stabilizeproteins during lyophilization.

In another embodiment of the present invention, the stabilizer may be anon proteinaceous stabilizing agent comprising a hyaluronic acid or apolyvinylpyrrolidone (KOLLIDON®, polyvinylpyrrolidone), hydroxyethylstarch, alginate or a polyethylene glycol or any combination thereof,such composition being optionally pH stabilized by a suitable pH buffer,in particular by a sodium acetate buffer, or a cryoprotectant or both.Said composition may comprise in addition to the mentioned stabilizerswater and at least one polyalcohol, such as mannitol or sorbitol ormixtures thereof. It may also comprise mono-, di- or higherpolysaccharides, such as glucose, sucrose or fructose. Such compositionis considered to be a safer composition possessing remarkable stability.

The hyaluronic acid in the instant pharmaceutical composition is in oneembodiment combined with the polypeptide of the invention in a quantityof 0.1 to 10 mg, especially 1 mg hyaluronic acid per ml in a 200 U/mlbotulinum toxin solution.

The polyvinylpyrrolidone (KOLLIDON®) when present in the instantcomposition, is combined with the polypeptide of the invention in such aquantity to provide a reconstituted solution comprising 10 to 500 mg,especially 100 mg polyvinylpyrrolidone per ml in a 200 U/ml polypeptideof the invention solution. In another embodiment reconstitution iscarried out in up to 8 ml solution. This results in concentrations ofdown to 12.5 mg polyvinylpyrrolidone per ml in a 25 U/ml polypeptide ofthe invention solution.

The polyethyleneglycol in the instant pharmaceutical composition is inone embodiment combined with the polypeptide of the invention in aquantity of 10 to 500 mg, especially 100 mg polyethyleneglycol per ml ina 200 U/ml botulinum toxin solution. In another embodiment, the subjectsolution also contains a 1-100 mM, in yet another embodiment 10 mMsodium acetate buffer.

The pharmaceutical composition in accordance with the present inventionin one embodiment retains its potency substantially unchanged for sixmonth, one year, two year, three year and/or four year periods whenstored at a temperature between about +8° C. and about −20° C.Additionally, the indicated pharmaceutical compositions may have apotency or percent recovery of between about 20% and about 100% uponreconstitution.

“Cryoprotectant” refers to excipients which result in an activeingredient, i.e. the polypeptide of the invention in a reconstituted oraqueous solution pharmaceutical composition that has greater than about20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, and up to about 100% of thetoxicity that the biologically active polypeptide of the invention hadprior to being freeze-dried in the pharmaceutical composition.

In another embodiment, the composition may contain a polyhydroxycompound, e.g. a polyalcohol as cryoprotectant. Examples of polyalcoholsthat might be used include, e.g., inositol, mannitol and othernon-reducing alcohols. Some embodiments of the composition do notcomprise a proteinaceous stabilizer, or do not contain trehalose ormaltotriose or lactose or sucrose or related sugar or carbohydratecompounds which are sometimes used as cryoprotectants.

The terms “preservative” and “preservatives” refer to a substance or agroup of substances, respectively, which prevent the growth or survivalof microorganisms, insects, bacteria or other contaminating organismswithin said composition. Preservatives also prevent said compositionfrom undesired chemical changes. Preservatives which can be used in thescope of this patent are all preservatives of the state of the art knownto the skilled person. Examples of preservatives that might be usedinclude, inter alia, e.g. benzylic alcohol, benzoic acid, benzalkoniumchloride, calcium propionate, sodium nitrate, sodium nitrite, sulphites(sulfur dioxide, sodium bisulfite, potassium hydrogen sulfite, etc.),disodium EDTA, formaldehyde, glutaraldehyde, diatomaceous earth,ethanol, methyl chloroisothiazolinone, butylated hydroxyanisole and/orbutylated hydroxytoluene.

The term “analgesic” relates to analgesic drugs that act in various wayson the peripheral and central nervous systems and includes inter aliaPARACETAMOL® (PARACETAMOL®, acetaminophen), the nonsteroidalanti-inflammatory drugs (NSAIDs) such as the salicylates, narcotic drugssuch as morphine, synthetic drugs with narcotic properties such asTRAMADOL® (TRAMADOL®, an opioid analgesics), and various others. Alsoincluded is any compound with a local analgesic effect such as e.g.lidocaine, benzylic alcohol, benzoic acid and others.

In one embodiment the analgesic is part of the composition, in anotherembodiment, the analgesic is administered before, during or after thetreatment with the chemodenervating agent.

The term “lyophilization” is used in this document for a treatment of asolution containing the polypeptide of the invention, whereas thissolution is frozen and dried until only the solid components of thecomposition are left over. The freeze-dried product of this treatment istherefore defined in this document as “lyophilisate”.

In this document the term “reconstitution” is defined as the process ofsolubilization of said freeze-dried composition of the polypeptide ofthe invention. This can be done by adding the appropriate amount ofsterile water, e.g. if all necessary components are already contained inthe lyophilisate. Or, if this is not the case, it can be done e.g. byadding a sterile saline-solution alone or if applicable with theaddition of components comprising e.g. a pH buffer, excipient,cryoprotectant, preservative, analgesic stabilizer or any combinationthereof. The saline of before mentioned “saline-solution” is asalt-solution, e.g. a sodium-chloride (NaCl) solution, or an isotonicsodium-chloride solution (i.e. a sodium-chloride concentration of 0.9%).The solubilization is carried out in such a manner that the final“reconstitution” is directly or indirectly, i.e. for example afterdilution, administrable to the patient. The neurotoxin may bereconstituted in isotonic media, e.g. in isotonic saline or sterilesaline.

It is noteworthy that the concept of the present invention, whichinvolves the administration of the polypeptide of the invention, for thetreatment of any condition which is associated with hyperactivecholinergic innervation of a muscle or an exocrine gland, where thepolypeptide of the invention blocks acetylcholine secretion into thesynaptic cleft. Therefore, treatment offered by the present inventionmay be directed at any of the following indications, most of which aredescribed in detail in Dressler D (2000) (Botulinum Toxin Therapy.Thieme Verlag, Stuttgart, N.Y.):

-   -   dystonia    -   cranial dystonia        -   blepharospasm        -   oromandibular dystonia            -   jaw opening type            -   jaw closing type        -   bruxism        -   Meige syndrome        -   lingual dystonia        -   apraxia of eyelid opening    -   cervical dystonia        -   antecollis        -   retrocollis        -   laterocollis        -   torticollis    -   pharyngeal dystonia    -   laryngeal dystonia        -   spasmodic dysphonia/adductor type        -   spasmodic dysphonia/abductor type        -   spasmodic dyspnea    -   limb dystonia        -   arm dystonia            -   task specific dystonia                -   writer's cramp                -   musician's cramps                -   golfer's cramp        -   leg dystonia            -   thigh adduction, thigh abduction            -   knee flexion, knee extension            -   ankle flexion, ankle extension            -   equinovarus deformity        -   foot dystonia            -   striatal toe            -   toe flexion            -   toe extension        -   axial dystonia            -   pisa syndrome            -   belly dancer dystonia        -   segmental dystonia        -   hemidystonia        -   generalised dystonia    -   dystonia in lubag    -   dystonia in corticobasal degeneration    -   dystonia in lubag    -   tardive dystonia    -   dystonia in spinocerebellar ataxia    -   dystonia in Parkinson's disease    -   dystonia in Huntington's disease    -   dystonia in Hallervorden Spatz disease    -   dopa-induced dyskinesias/dopa-induced dystonia    -   tardive dyskinesias/tardive dystonia    -   paroxysmal dyskinesias/dystonias        -   kinesiogenic        -   non-kinesiogenic        -   action-induced    -   palatal myoclonus    -   myoclonus    -   myokymia    -   rigidity    -   benign muscle cramps    -   hereditary chin trembling    -   paradoxic jaw muscle activity    -   hemimasticatory spasms    -   hypertrophic branchial myopathy    -   maseteric hypertrophy    -   tibialis anterior hypertrophy    -   nystagmus    -   oscillopsia    -   hyperhydrosis    -   supranuclear gaze palsy    -   epilepsia partialis continua    -   planning of spasmodic torticollis operation    -   abductor vocal cord paralysis    -   recalcitant mutational dysphonia    -   upper oesophageal sphincter dysfunction    -   vocal fold granuloma    -   stuttering    -   Gilles de la Tourette syndrom    -   middle ear myoclonus    -   protective larynx closure    -   postlaryngectomy speech failure    -   protective ptosis    -   entropion    -   sphincter Odii dysfunction    -   pseudoachalasia    -   nonachalsia oesophageal motor disorders    -   vaginismus    -   postoperative immobilisation    -   tremor    -   genito-urinary diseases        -   bladder dysfunction        -   overactive bladder            -   urinary incontinence            -   urinary retention            -   spastic bladder    -   gastro-intestinal diseases    -   detrusor sphincter dyssynergia    -   bladder sphincter spasm    -   hemifacial spasm    -   reinnervation dyskinesias    -   cosmetic use    -   crow's feet        -   frowning        -   facial asymmetries        -   mentalis dimples        -   glabella frown line        -   frontal lines        -   platysma        -   smoker's lines        -   marionette lines        -   masseter lift    -   stiff person syndrome    -   tetanus    -   prostate diseases        -   prostate hyperplasia        -   prostate cancer    -   adipositas treatment    -   infantile cerebral palsy    -   strabismus    -   mixed    -   paralytic    -   concomitant    -   after retinal detachment surgery    -   after cataract surgery    -   in aphakia    -   myositic strabismus    -   myopathic strabismus    -   dissociated vertical deviation    -   as an adjunct to strabismus surgery    -   esotropia    -   exotropia    -   achalasia    -   anal fissures    -   exocrine gland hyperactivity    -   Frey syndrome    -   Crocodile Tears syndrome    -   hyperhidrosis        -   axillar        -   palmar        -   plantar    -   rhinorrhea    -   relative hypersalivation        -   in stroke        -   in parkinsosn's        -   in amyotrophic lateral sclerosis    -   spastic conditions        -   in encephalitis and myelitis            -   autoimmune processes                -   multiple sclerosis                -   transverse myelitis                -   Devic syndrome            -   viral infections            -   bacterial infections            -   parasitic infections            -   fungal infections        -   in hereditary spastic paraparesis        -   postapoplectic syndrome            -   hemispheric infarction            -   brainstem infarction            -   myelon infarction        -   in central nervous system trauma            -   hemispheric lesions            -   brainstem lesions            -   myelon lesion        -   in central nervous system hemorrhage            -   intracerebral hemorrhage            -   subarachnoidal hemorrhage            -   subdural hemorrhage            -   intraspinal hemorrhage        -   in neoplasias            -   hemispheric tumors            -   brainstem tumors            -   myelon tumors    -   headache        -   migraine        -   tension headache        -   sinus headache        -   chronic headache    -   and/or hair loss.

The pharmaceutical composition comprising the botulinum toxin isadministered, in one embodiment several times, in an effective amountfor improving the patient's condition. It also has to be noted thatdepending on the persistency of the polypeptide of the invention loweror higher dosages are needed, therefore, the following dosing referencesare just for orientation purpose.

Typically, the dose administered to the patient will be up to about 1000units, but in general should not exceed 400 units per patient. In oneembodiment the range lies between about 80 to about 400 units. Thesevalues are in one embodiment valid for adult patients. For children, therespective doses range from 25 to 800 and in another embodiment from 50to 400 units.

While the above ranges relate to the maximum total doses, the dose rangeper muscle is in one embodiment within 3 to 6 units/kg body weight(b.w.), for small muscles 0.5-2 U/kg b.w., in another embodiment 0.1-1U/kg b.w. Generally doses should not exceed 50 Upper injection site and100 U per muscle.

In one embodiment of the present invention the effective amount ofbotulinum toxin administered exceeds 500 U of polypeptide of theinvention in adults or exceeds 15 U/kg body weight in children.

As to the frequency of dosing, the re-injection interval depend greatlyon the persistency of the modified neurotoxin. Thus, according to thepresent invention the medicament to be administered is re-administeredin intervals of between 3 and 6 months, in another embodiment themedicament is re-administered in intervals of between 2 weeks and lessthan 3 months. However, depending on the modifications of theneurotoxin, in other embodiments treatments of more than 6 months up to12 months or treatments in time periods shorter than 2 weeks areenvisaged.

With regard to the composition and dosing of the medicament on the basisof botulinum toxin, and in regard to the composition, dosing andfrequency of administration of the medicament on the basis of theneurotoxic component of botulinum toxin, U.S. 60/817,756 is incorporatedherein by reference.

While the above stated values are to be understood as a generalguideline for administering the medicament as used within the presentinvention, it is, however, ultimately the physician who is responsiblefor the treatment who decides on both the quantity of toxin administeredand the frequency of its administration.

The medicament on the basis of botulinum toxin can be injected directlyinto the affected muscles. In order to find the appropriate injectionsite, several means exist which help the physician in order to find thesame. Within the present invention, all methods for finding the bestinjection site are applicable, such as injection guided byelectromyography (EMG), injection guided by palpation, injection guidedby CT/MRI, as well as injection guided by sonography (ultra-sound).Among those methods, the latter is in one embodiment the method ofchoice when treating children. With respect to further details regardingthe injection guided by sonography, we refer to Berweck“Sonography-guided injection of botulinum toxin A in children withcerebral palsy”, Neuropediatric 2002 (33), 221-223.

The term “injection” is defined as any process, which allows the personskilled in the art to administer the active agent to the target site bypenetrating the skin. An incomplete number of examples for “injections”are subcutaneous, intra-muscular, intra-venous, intra-thecal,intra-arterial, etc.

It is to be understood that the terminology used herein is for thepurpose of describing particular embodiments only and is not intended tobe limiting. It must be noted that, as used in the specification and theappended claims, the singular forms “a”, “an” and “the” include pluralreferents unless the context clearly dictates otherwise.

Literature:

de Paiva A, Meunier F A, Molgo J, Aoki K R, Dolly J O. Related Articles,Functional repair of motor endplates after botulinum neurotoxin type Apoisoning: biphasic switch of synaptic activity between nerve sproutsand their parent terminals. Proc Natl Acad Sci USA. 1999; 96(6):3200-5.

E. L. V. Harris (Ed.), S. Angal (Ed.), “Protein Purification Methods: APractical Approach”, Oxford University Press (December 1989), ISBN-10:019963002X, ISBN-13: 978-0199630028

Eleopra R, Tugnoli V, Quatrale R, Gastaldo E, Rossetto O, De Grandis D,Montecucco C. Botulinum neurotoxin serotypes A and C do not affect motorunits survival in humans: an electrophysiological study by motor unitscounting. Clin Neurophysiol. 2002; 113(8):1258-64.

Eleopra R, Tugnoli V, Rossetto O, Montecucco C, De Grandis D. Botulinumneurotoxin serotype C: a novel effective botulinum toxin therapy inhuman. Neurosci Lett. 1997; 224(2):91-4.

Foran P G, Mohammed N, Lisk G O, Nagwaney S, Lawrence G W, Johnson E,Smith L, Aoki K R, Dolly J O. Evaluation of the therapeutic usefulnessof botulinum neurotoxin B, C1, E, and F compared with the long lastingtype A. Basis for distinct durations of inhibition of exocytosis incentral neurons. J Biol. Chem. 2003 Jan. 10; 278(2):1363-71. [Epub 2002Oct. 14]

John M. Walker, Humana Press; “The Protein Protocols Handbook (Methodsin Molecular Biology)”, Volume: 2 (February 2002), ISBN-10: 0896039404,ISBN-13: 978-0896039407

Jurasinski C V, Lieth E, Dang Do A N, Schengrund C L Correlation ofcleavage of SNAP-25 with muscle function in a rat model of Botulinumneurotoxin type A induced paralysis Toxicon. 2001; 39(9):1309-15

Robert K. Scopes, “Protein Purification: Principles and Practice”,Verlag: Springer, Berlin; Auflage: 3 Sub (Januar 1994), ISBN-10:0387940723, ISBN-13: 978-0387940724

The present invention is now further exemplified by way of thenon-limited examples recited herein under.

EXAMPLES Example 1 Construction of an Expression Plasmid

The DNA-sequence of the heavy chain of botulinum toxin A is amplifiedfrom chomosomal DNA of C. botulinum Type A (database No. AAA23262) byPCR. At the 5′ end a sequence is added coding for the recognitionsequence of thrombin. At the 3′ end a DNA sequence is added, coding foran affinity tag peptide, suitable for later purification (e.g. His-tagor Strep-tag). The DNA is inserted into an expression plasmid. The DNAsequences for the first and second light chain are also of serotype Aand are amplified in a similar fashion from chromosomal DNA of C.botulinum type A (database No. AAA23262) via PCR. The sequence of thelight chain is then introduced twice consecutively in the expressionplasmid upstream of the thrombin recognition sequence (TE). In totaltherefore the sequences shows the following coding structure:LC-LC-TE-HC-Tag.

Example 2 Production of the Fusion Protein in E. Coli

The fusion protein is transfected into E.coli TG1. The induction isperformed at 21° C. for 4 hours. The fusion protein is then purified via(STREP-TACTIN® SEPHAROSE® (STREP-TACTIN® SEPHAROSE®, agarose-basedmatrix for purification of recombinant Strep-tagged proteins; IBA GmbH,Göttingen) column chromatography according to manufacturers protocol.The fusion protein then is activated by immobilized thrombin (ThrombinSEPHAROSE® agarose-based matrix) which cleaves the peptide linkagebetween the heavy chain and the two light chains. The subunits of theprotein remain only connected via disulfide bonds.

Example 3 Test of Persistency (Extensor Digitorum Brevis, EDB)

A test person is applied with 4 units Xeomin® (Merz PharmaceuticalsGmbH) into the right EDB solubilized in 0.1 ml physiological saline andinto the left EDB 4 units of a modified botulinum toxin (fusion proteinof botulinum toxin type A conjugated with an additional light chain ofbotulinum toxin type A). Each 30 days the “compound muscle actionpotential” (CMAP) is electrophysiological measured. After 90 days theamplitude of the CMAP of the right EDB is reduced about 40% (incomparison to the starting activity) whereas in the EDB on the left sidethe amplitude is reduced at about 70%. On the left side the CMAP reaches40% after 150 days.

Example 4 Prolongation of Persistency

A patient suffering from torticollis spasmodicus is treated with BOTOX®(Allergen, Inc.) (240 units). He has to be treated every 10 to 12 weeksin a neurological office due to a decreased activity of the botulinumtoxin, The patient then receives an injection of 240 units of a modifiedneurotoxin (botulinum toxin type A with an additional fused light chainof botulinum toxin type A). The patient needs no further injection till18 weeks after the first treatment.

The invention claimed is:
 1. A nucleic acid encoding a polypeptidecomprising: (a) a heavy chain (HC) domain of a neurotoxic component of aclostridial toxin; (b) a first light chain (LC) domain of a neurotoxiccomponent of a clostridial toxin; and (c) at least one further LC domainof a neurotoxic component of a clostridial toxin; wherein the domainsare connected by a direct linkage via a covalent bond, a peptide-linker,a chemical linker, or a combination of two or more thereof, and whereinthe first and at least one further LC domains may be the same ordifferent from each other, and wherein each of the first and at leastone further LC domains exhibit proteolytic activity.
 2. A vectorcomprising the nucleic acid of claim
 1. 3. A host cell comprising thenucleic acid of claim
 1. 4. A host cell comprising the vector of claim2.
 5. A method for producing a polypeptide comprising the steps ofcultivating the host cell of claim 4, producing and purifying apolypeptide encoded by the nucleic acid comprised in the vector and,optionally, formulating the polypeptide in a pharmaceutical composition.